BACKGROUND OF THE INVENTION
[0001] The present invention relates to a non-invasive method and apparatus for detecting
biological activities in a fluid specimen, such as blood, urine or sputum, where the
specimen and a culture medium are introduced into sealable containers and are exposed
to conditions enabling a variety of metabolic, physical, and chemical changes to take
place in the presence of microorganisms in the sample. The biological activity being
detected by a variety of chemical sensors that are based on changes in fluorescence
lifetime and/or intensity.
[0002] Usually, the presence of microorganisms such as bacteria in a patient's body fluid,
particularly blood, is determined using blood culture vials. A small quantity of blood
is injected through a sealing rubber septum into a sterile vial containing a culture
medium. The vial is incubated at a temperature conducive to bacterial growth, e.g.,
37°C, and monitored for such growth.
[0003] Common visual inspection involves monitoring the turbidity of the liquid suspension.
Known instrumental methods detect changes in the CO₂ content in the headspace of the
culture bottles, which is a metabolic by-product of the bacterial growth. Monitoring
the CO₂ content can be accomplished by conventional methods, including radiochemical,
infrared absorption at a CO₂ spectral line, or pressure/vacuum measurement. These
methods, however, require invasive procedures which can result in cross-contamination
between vials.
[0004] Recently, novel non-invasive methods have been developed which use chemical sensors
inside a vial. Such sensors often respond to changes in the CO₂ concentration by changing
their color or by changing their fluorescence intensity. The outputs from these sensors
are based upon light intensity measurements. This means that errors may occur, particularly
if the light sources used to excite the sensors, or the photodetectors used to monitor
intensities, exhibit aging effects over time.
[0005] In known automated non-invasive blood culture systems, individual light sources,
individual spectral excitation and emission filters, and individual photodetectors
are arranged adjacent to each vial. Such arrangements result in certain station sensitivity
variations from one vial to the next. Due to the fact that most known blood culture
sensors generate only a moderate contrast ratio in the measured photocurrent during
bacterial growth, extensive and time-consuming calibration procedures and sophisticated
detection algorithms are required to operate these systems. Moreover, light sources,
spectral filters, and photodetectors with extreme narrow specification tolerances
must be utilized.
[0006] The disadvantage of such intensity-based sensor arrangements can be overcome by utilizing
fluorescent sensors that change their fluorescence lifetime, wherein intensity measurement
is replaced with time parameter measurement and intensity changes have no impact on
the sensor output signal. Many chemical sensor materials are known that change their
fluorescence lifetime with changing oxygen concentration, pH, carbon dioxide concentration,
or other chemical parameters (see, e.g., G.B. Patent No. 2,132,348).
[0007] A change in sensor fluorescence lifetime is commonly monitored by applying a well-known
phase shift method (see, e.g., U.S. Patent No. 5,030,420), wherein the excitation
light is intensity-modulated. That method results in an intensity-modulated fluorescence
emission that is phase-shifted relative to the excitation phase. Phase shift angle,
ϑ, being dependent on the fluorescence lifetime, τ, according to the equation:

where ω = 2πf, is the circular light modulation frequency.
[0008] An inspection of equation (1) reveals that the phase shift method allows for maximum
resolution, dϑ/dτ, under the condition ωτ = 1. Unfortunately, almost all known pH-
or carbon dioxide-sensitive fluorophores have decay times in the range 5 ns to 500
ps. In other words, light modulation frequencies, f = 1/2πτ, in the range 32 MHz to
320 MHz would be required.
[0009] It is possible to accomplish light intensity modulation at such high frequencies,
however, this would require acousto-optic or electro-optic modulators which are only
efficient in combination with lasers. Moreover, detecting the modulated fluorescence
light would require highly sensitive high-speed photodetectors, such as microchannel-plate
photomultipliers, which are rather expensive. Consequently, all commercial automated
blood culture systems are based on intensity monitoring, and none utilize time-resolved
fluorescent carbon dioxide sensors.
SUMMARY OF THE INVENTION
[0010] The present invention overcomes problems identified in the art by providing a method
and apparatus for reliably and non-invasively detecting biological activities in blood
culture vials that do not have the fluorescence intensity limitations discussed above.
[0011] According to the present invention, a culture medium and blood specimen are introduced
into a sealable glass vial having a headspace gas mixture such that a change in the
gas mixture composition can be monitored by a chemically sensitive composite material
in the vial. The chemically sensitive composite material comprises a mixture of a
fluorophore and a chromophore. The fluorophore exhibits a long fluorescence decay
time and a fluorescence intensity that depend on a first chemical parameter, such
as oxygen concentration. The chromophore exhibits an optical transmission that depends
on a second chemical parameter, such as carbon dioxide concentration, the optical
transmission of the chromophore changing with the second chemical parameter either
within the excitation or within the emission wavelength range of the fluorophore.
[0012] By illuminating the composite sensor matrix with intensity-modulated light, measuring
the AC component and the DC component of the fluorescence photocurrent separately,
and by processing these signals in a computer, a sensor output signal is produced
that shows a significantly increased contrast ratio, compared with known optical blood
culture sensors. In addition, the composite sensor allows for separation of the oxygen
consumption effect and the carbon dioxide production effect, and requires only a relatively
low light modulation frequency (150 kHz). Therefore, a low-cost light emitting diode
(LED) can be used as the excitation source.
[0013] More particularly, the fluorophore and the chromophore are mixed into the same sensor
matrix and are illuminated with intensity-modulated excitation light. The modulation
frequency is chosen so that the condition ωτ = 1 holds for the fluorophore when the
fluorescence lifetime has its minimum value. The fluorescence light emitted by the
composite sensor is monitored using only one photodetector. The fluorescence photocurrent
from the photodetector is split into its AC and DC components, that are measured separately.
A sensor output signal is then generated by dividing the measured DC component by
the calculated fluorescence modulation degree, which is equal to the AC/DC ratio of
the components.
[0014] In an aerobic vial, bacterial growth causes at first a decrease in the oxygen concentration.
This, in turn, results in an increase in the fluorescence intensity and in the fluorescence
lifetime of the fluorophore. If the light modulation frequency for the excitation
source is selected properly, the increase in lifetime causes a decrease in the fluorescence
modulation degree. As soon as carbon dioxide is produced by the bacteria, the chromophore's
optical transmission increases, which results in a further amplification in the emitted
fluorescence intensity. In a sensor arrangement according to the present invention,
the combined effects of oxygen consumption and carbon dioxide production generate
a significantly higher change in the measured fluorescence photocurrent than in known
blood culture sensors.
[0015] As mentioned above, the fluorescence modulation degree is also calculated. The modulation
degree can be measured at very high precision because all artifacts due to light source
aging, optical filter variations, vial displacement, and photodetector sensitivity
changes are canceled out. Therefore, it is practical to utilize the modulation degree
to calculate a final sensor output signal. This final signal is obtained by dividing
the increasing DC component by the decreasing modulation degree. Due to the opposite
trends of these two quantities, a further amplification effect results with regard
to the final sensor output signal. Consequently, the sensor output signal shows a
contrast ratio due to bacterial growth that is larger by almost two orders of magnitude
as compared to known blood culture sensors. This allows for better detection, and
the requirements with regard to part's tolerances are reduced.
[0016] In general, a time delay is observed between oxygen consumption and carbon dioxide
production. Therefore, analyzing the final sensor output signal allows for the separation
of the two effects. By taking into account the magnitude, speed, time of occurrence
and relative time delay between the two mechanisms (oxygen and carbon dioxide), information
regarding the microorganism species can be gained. Moreover, even if no time delay
should occur, the two mechanisms can be resolved by analyzing the fluorescence modulation
degree, as explained below.
[0017] These and other features, objects, benefits and advantages of the present invention
will become more apparent upon reading the following detailed description of the preferred
embodiments, along with the appended claims in conjunction with the drawings, wherein
reference numerals identify corresponding components.
DESCRIPTION OF THE DRAWINGS
[0018]
Fig. 1 shows schematically a composite optical blood culture sensor arrangement according
to the present invention;
Fig. 2 is a plot showing fluorescence intensity versus time for the fluorophore, in
response to oxygen consumption by the microorganisms.
Fig. 3 is a plot showing frequency lifetime product, ωτ, versus time for the fluorophore,
in response to oxygen consumption by the microorganisms;
Fig. 4 is a plot showing fluorescence intensity versus time in response to carbon
dioxide production;
Fig. 5 is a plot showing modulation degree, AC/DC, of the fluorescence emission versus
time in response to oxygen consumption and carbon dioxide production;
Fig. 6 is a plot showing fluorescence intensity versus time in response to oxygen
consumption and delayed carbon dioxide production;
Fig. 7 is a plot showing fluorescence intensity divided by modulation degree versus
time in response to oxygen consumption and delayed carbon dioxide production;
Fig. 8 is a plot showing the first derivative of the signal shown in Fig. 7;
Fig. 9 is a plot showing fluorescence intensity versus time, like the plot shown in
Fig. 4, but for a weak carbon dioxide producer;
Fig. 10 is a plot showing fluorescence intensity divided by modulation degree versus
time, like the plot shown in Fig. 7, but for a weak carbon dioxide producer.
Fig. 11 is a plot showing the first derivative of the signal shown in Fig. 10, for
the weak carbon dioxide producer;
Fig. 12 is a plot showing standard spectral transmission characteristics for a pH/carbon
dioxide-sensitive chromophore;
Fig. 13 is a plot showing modulation degree of the fluorescence emission for an oxygen
sensor; and
Fig. 14 is a plot showing the first derivative of a growth curve and its characteristic
features.
DETAILED DESCRIPTION
[0019] A preferred embodiment of a composite optical blood culture sensor arrangement embodying
the principles and concepts of the invention is depicted schematically in Fig. 1.
In this arrangement, a specimen and culture medium mixture 4 are introduced into an
optically transparent container 1 that is sealed by a cap 2. A mixture of chemical
sensor materials 3 is disposed to an inner wall 20 or an inner bottom surface 21 of
container 1. The mixture 3 is illuminated by an excitation light source 5, preferably
a blue LED 5, that is connected to an electronic signal source 7. Signal source 7
provides a DC bias and a high-frequency modulation voltage to light source 5 over
a line 30, and is equipped with a power control input 25 connected by a line 31 to
a computer 12.
[0020] Sensor material mixture 3 comprises a mixture of a fluorophore and a chromophore,
wherein the fluorophore exhibits a long fluorescence decay time and a fluorescence
intensity that depend on a first chemical parameter, such as oxygen concentration.
The chromophore, however, exhibits an optical transmission that depends on a second
chemical parameter, such as carbon dioxide concentration. The optical transmission
of the chromophore changing with the second chemical parameter, either within the
excitation or within the emission wavelength range of the fluorophore.
[0021] Fluorescence light reemerging from sensor material mixture 3 is detected by a photodetector
9, e.g., a photomultiplier. An emission filter 8 is arranged between mixture 3 and
photodetector 9, and an excitation filter 6 is mounted in front of light source 5,
to prevent excitation light from light source 5 reaching photodetector 9. Photodetector
9 generates an output signal on a line 35 that is fed to a power splitter 10. Power
splitter 10 then generates two output signals, one of which is connected by a line
40 to the input of a low-pass filter 11 that is connected directly to computer 12.
The other output signal of power splitter 10 is fed by a line 45 to the input of a
band-pass filter 13 that is connected via a high-frequency voltmeter 14 to computer
12. Computer 12 is connected to a data display unit 15 to display information.
[0022] In operation, light source 5 illuminates sensor material mixture 3 with excitation
light that is intensity-modulated at a circular modulation frequency, ω, having a
modulation degree, m. The emitted fluorescence intensity of sensor material mixture
3 can be described by:

with T(t) being the carbon dioxide-dependent optical transmission of the chromophore,
F₀(t) being the oxygen-dependent average fluorescence intensity, and

being the fluorescence phase shift relative to the excitation modulation phase. In
equation (2), m represents the modulation degree of the excitation light, which can
be as high as 1. For all plots that follow, a constant source modulation degree, m
= 0.7, a very reasonable value, is assumed. The quantity τ(t) in equations (2) and
(3) is fluorescence lifetime.
[0023] Average fluorescence intensity, F₀(t), may depend on time, t, according to the expression:

where k₁ is a constant, and h(t) is a time-dependent function that rises from a first,
lower level to a second, higher level as a consequence of oxygen consumption during
microorganism growth. Fig. 2 is a plot showing fluorescence intensity versus time
for a fluorophore, in response to oxygen consumption by the microorganisms.
[0024] Fluorescence lifetime τ(t), may depend on time, t, according to the expression:

where k₂ is a constant, and h(t) is the same time-dependent function that rises from
a first, lower level to a second, higher level as a consequence of oxygen consumption
during microorganism growth.
[0025] Modulation frequency, ω, is chosen so that the condition ωτ ≈ 1 holds for the fluorophore
when it has its minimum τ-value. Fig. 3 is a plot showing frequency lifetime product,
ωτ, versus time for the fluorophore, in response to oxygen consumption by the microorganisms.
The light modulation frequency is selected so that ωτ = 1 prior to oxygen consumption
if the fluorescence lifetime is short.
[0026] Optical transmission of the chromophore, T(t), is also a time-dependent function
that rises from a first, lower level to a second, higher level as a consequence of
carbon dioxide production during microorganism growth. Fig. 4 is a plot showing the
corresponding increase in the remitted fluorescence intensity versus time in response
to carbon dioxide production. The carbon dioxide response exhibits a time delay relative
to oxygen response.
[0027] In a composite optical blood culture sensor arrangement according to the present
invention, the fluorescence photocurrent, I(t), is given by the expression:

where K is a constant. Photocurrent I(t) is then split into AC and DC components that
are measured separately, so that an AC/DC ratio can then be calculated within computer
12. Based on this, we obtain for the AC component:

for the DC component:

and for the fluorescence modulation degree:

From equation (9), it can be seen that fluorescence modulation degree, AC/DC, does
not change with a change in carbon dioxide concentration. This is illustrated in Fig.
5, where modulation degree, AC/DC, of the re-emitted fluorescence is plotted versus
time, in response to oxygen consumption and carbon dioxide production. Equation (9)
also shows that modulation degree, AC/DC, is independent of K, therefore, almost free
from instrumental artifacts.
[0028] Fig. 6 is a plot of the DC signal, i.e., fluorescence intensity versus time in response
to oxygen consumption and delayed carbon dioxide production. The contrast ratio due
to the combined oxygen and carbon dioxide effect is 19.1, which is the product of
the carbon dioxide contrast ratio of 2.25 and the oxygen contrast ratio of 8.5.
[0029] Fig. 7 is a plot showing fluorescence intensity, DC, divided by modulation degree,
AC/DC, versus time in response to oxygen consumption and delayed carbon dioxide production.
The plot in Fig. 7 represents a final sensor output signal that is displayed by computer
12 on display 15. It should be appreciated that the contrast ratio, relative to the
contrast ratio in Fig. 6, is increased further from 19.1 to a value of 120.
[0030] Fig. 8 is a plot showing a first derivative of the signal in Fig. 7, and illustrates
that the effects of oxygen consumption and carbon dioxide production are clearly separated.
In this case, both peaks exhibit comparable heights.
[0031] Applicant has also found that the effects of oxygen consumption and carbon dioxide
production can also be resolved, if no time delay should occur. This is possible simply
by analyzing the final sensor output signal
and the modulation degree, AC/DC. While final sensor output responds to both effects,
modulation degree responds only to the oxygen effect. Therefore, it is possible to
separate each effect.
[0032] Fig. 9 corresponds to Fig. 4, but shows a plot of fluorescence intensity versus time
for a weak carbon dioxide producer; Fig. 10 corresponds to Fig. 7, but shows the final
sensor output signal expected for a weak carbon dioxide producer; and Fig. 11 corresponds
to Fig. 8, but shows the differentiated signal expected for a weak carbon dioxide
producer. Again, the effects of oxygen consumption and carbon dioxide production are
clearly separated, but now the two peaks exhibit very different heights.
[0033] Fig. 12 is a plot showing a typical spectral transmission characteristic for a pH/carbon
dioxide-sensitive chromophore. The family of curves shown in Fig. 12 illustrate the
change in optical transmission with changing chemical input parameter, the indicated
excitation range corresponding to the emission wavelength range of blue SiC light-emitting
diodes. The indicated emission range corresponds to the emission wavelength range
of available fluorescent oxygen sensors.
[0034] Fig. 13 shows the modulation degree, AC/DC, of a fluorescence emission for an oxygen
sensor, the light modulation frequency having been set to 140 kHz. In this case, the
condition ωτ = 1 is fulfilled for an oxygen concentration of 11%. In doing so, changes
in the initial high oxygen concentration have only a minor effect on the measured
modulation degree. This is of advantage, in view of the required long shelf life of
typical blood culture vials. In general, an optimization is possible by selecting
a particular light modulation frequency.
[0035] Fig. 14 is a plot showing the first derivative of a growth curve and its characteristic
features. In Fig. 14, T1 and T2 are the times of occurrence for the oxygen and the
carbon dioxide effect. Parameters t1 and t2 indicate the duration for each of these
effects and parameters A1 and A2 are related to the strength of both effects. Applicant
has found that by compiling a set of these six characteristic parameters for each
sample vial and comparing the set with the corresponding data base that has been generated
using known samples at an earlier time, a presumptive microorganism identification
can be achieved.
[0036] It should be understood that the above-described embodiment is simply illustrative
of an apparatus embodying the principles and concepts of the present invention. For
example, the mixture of chemical sensor materials 3, shown in Fig. 1, disposed to
the wall or bottom surface of container 1 could be replaced with a bi-layer structure
having the fluorophore and chromophore contained in two adjacent layers rather than
mixed together. The chromophore would be attached first to container 1 and the fluorophore
would then be added as a second top layer. While this may be a little more expensive
because of the two production steps required, the effect of changing chromophore transmission
on the emitted fluorescence would be even stronger, since every fluorescence excitation
and/or emission photon would have to traverse the full chromophore layer. In the mixed
option, shown in Fig. 1, some photons never "meet" a chromophore molecule, but only
meet a fluorophore molecule to generate a new fluorescence photon and therefore leave
the sensor material without interacting with any chromophore molecule. Of course,
other suitable variations and modifications could also be made to the apparatus described
and still remain within the scope of the present invention.
1. An apparatus for detecting microorganism growth comprising:
a container comprising a culture medium, a blood specimen, and a headspace having
a concentration of a gas;
a chemically sensitive material in said container for detecting microorganism growth
within said container when illuminated with an intensity-modulated light, said chemically
sensitive material being comprised of a first material and a second material;
said first material exhibiting a fluorescence decay time and fluorescence intensity
that depend on a first chemical parameter of said gas; and
said second material exhibiting an optical transmission that depends on a second
chemical parameter of said gas.
2. An apparatus according to Claim 1, wherein said first chemical parameter of said gas
is oxygen concentration, such that said first material exhibits a change in fluorescence
decay time and fluorescence intensity in response to the change in oxygen concentration
of said gas.
3. An apparatus according to Claim 2, wherein said first material is a fluorophore.
4. An apparatus according to Claim 1, wherein said second chemical parameter of said
gas is carbon dioxide concentration, such that said second material exhibits a change
in optical transmission in response to the change in carbon dioxide concentration
of said gas.
5. An apparatus according to Claim 4, wherein said second material is a chromophore.
6. An apparatus according to Claim 4, wherein said second material exhibits a change
in optical transmission in response to the change in carbon dioxide concentration
of said gas within the excitation range of said first material.
7. An apparatus according to Claim 4, wherein said second material exhibits a change
in optical transmission in response to the change in carbon dioxide concentration
of said gas within the emission wavelength range of said first material.
8. An apparatus according to Claim 1, wherein:
said first material is a fluorophore that exhibits a change in fluorescence decay
time and fluorescence intensity in response to a change in oxygen concentration of
said gas; and
said second material is a chromophore that exhibits a change in optical transmission
in response to a change in carbon dioxide concentration of said gas,
whereby the change in fluorescence intensity emitted by said fluorophore is amplified
by the optical transmission change of said chromophore.
9. An apparatus according to Claim 1, further comprising:
a light source for illuminating said chemically sensitive material in said container
with excitation light that is intensity-modulated at a predetermined circular modulation
frequency with a predetermined modulation degree;
a photodetector for detecting fluorescence light reemerging from said chemically
sensitive material;
means for measuring an AC component and a DC component of the fluorescence photocurrent
from said photodetector; and
means for generating a sensor output signal based on the AC component and the DC
component representing information as to whether or not microorganism growth is present
within said container.
10. An apparatus according to Claim 1, further comprising:
means for compiling a first set of signals corresponding to said fluorescence decay
time and fluorescence intensity depending on said first chemical parameter of said
gas;
means for compiling a second set of signals corresponding to said optical transmission
depending on said second chemical parameter of said gas; and
means for comparing the first and second sets of signals with a corresponding predetermined
data base of first and second sets of signals for known microorganisms to perform
microorganism identification on a detected microorganism.
11. An apparatus for detecting microorganism growth comprising:
a container comprising a culture medium, a blood specimen, and a headspace having
a concentration of a gas;
a chemically sensitive material in said container for detecting microorganism growth
within said container when illuminated with an intensity-modulated light, said chemically
sensitive material being comprised of a first material and a second material;
said first material is a fluorophore that exhibits a change in fluorescence decay
time and fluorescence intensity in response to a change in oxygen concentration of
said gas;
said second material is a chromophore that exhibits a change in optical transmission
in response to a change in carbon dioxide concentration of said gas, whereby the change
in fluorescence intensity emitted by said fluorophore is amplified by the optical
transmission change of said chromophore;
a light source for illuminating said chemically sensitive material in said container
with the intensity-modulated light at a predetermined circular modulation frequency
and a predetermined modulation degree;
a photodetector for detecting fluorescence light reemerging from said chemically
sensitive material;
means for measuring an AC component and a DC component of the fluorescence photocurrent
from said photodetector; and
means for generating a sensor output signal based on the AC component and the DC
component representing information as to whether or not microorganism growth is present
within said container.